Intramedullary bone tissue reaction of ion-releasing resin-modified glass-ionomer restoration versus two calcium silicate-based cements: an animal study

This comparative study was conducted to assess the intramedullary bone tissue reaction of an ion-releasing resin modified glass-ionomer cement with claimed bioactivity (ACTIVA bioactive resin) restorative material versus Mineral Trioxide Aggregate High Plasticity (MTA HP) and bioceramic putty iRoot BP Plus. Fifty-six adult male Wistar rats were assigned into 4 equal groups (14 rats each). A surgical intramedullary bi-lateral tibial bone defects were performed in rats of the control group I (GI) and left without any treatment to be considered as controls (n = 28). The rats of groups II, III and IV were handled as group I except that the tibial bone defects were filled with ACTIVA, MTA HP and iRoot BP, respectively. In all groups, rats were euthanized after one month and specimens were processed to histological investigation, SEM examination and EDX elemental analysis. In addition, semi-quantitative histomorphometric scoring system was conducted for the following parameters; new bone formation, inflammatory response, angiogenesis, granulation tissue, osteoblasts and osteoclasts. The clinical follow-up outcome of this study revealed the recovery of rats after 4 days post-surgical procedure. It was observed that the animal subjects returned to their routine activities, e.g., walking, grooming and eating. The rats showed normal chewing efficiency without any weight loss or postoperative complications. Histologically, the control group sections showed scanty, very thin, new bone trabeculae of immature woven type located mostly at the peripheral part of the tibial bone defects. These defects exhibited greater amount of thick bands of typically organized granulation tissue with central and peripheral orientation. Meanwhile, bone defects of ACTIVA group showed an empty space surrounded by thick, newly formed, immature woven bone trabeculae. Moreover, bone defects of MTA HP group were partially filled with thick newly formed woven bone trabeculae with wide marrow spaces presented centrally and at the periphery with little amount of mature granulation tissue at the central part. The iRoot BP Plus group section exhibited an observable woven bone formation of normal trabecular structures with narrow marrow spaces presented centrally and at the periphery showed lesser amount of well-organized/mature granulation tissue formation. Kruskal Wallis test revealed total significant differences between the control, ACTIVA, MTAHP and iRoot BP Plus groups (p < 0.05). Meanwhile, Mann–Whitney U test showed significant difference between control and ACTIVA groups, Control and MTA HP groups, control and iRoot BP Plus groups. ACTIVA and MTA HP groups, ACTIVA and iRoot BP Plus (p ˂ 0.05) with no significant difference between MTA HP and iRoot BP Plus (p > 0.05). The elemental analysis outcome showed that the lesions of the control group specimens were filled with recently created trabecular bone with limited marrow spaces. EDX tests (Ca and P analysis) indicated a lower degree of mineralization. Lower amounts of Ca and P was expressed in the mapping analysis compared with other test groups. Calcium silicate-based cements induce more bone formation when compared to an ion-releasing resin modified glass-ionomer restoration with claimed bioactivity. Moreover, the bio-inductive properties of the three tested materials are likely the same. Clinical significance: bioactive resin composite can be used as a retrograde filling.


Materials and methods
Materials used for the present study are listed in Table 1. Surgical procedures. Anesthesia was performedafter intraperitoneal injection of ketamine hydrochloride and xylazine hydrochloride (3: 1 respectively; 0.05 mL/100 g of weight). Bilaterally, the fur leg was clipped, and operation site was wrapped in a sterile fashion with 0.12% chlorhexidine and the residual antiseptic was removed with sterile moistened gauze. A one cm longitudinal incision along the anteromedial border of tibia was performed using # 11 scalpel. The medial diaphyseal face of the tibia was exposed via blunt dissection using blunt scissor to expose a full mucoperiosteal flap with care taken during splitting the muscle to avoid injury to the tibial arterial and its venous return. A non-critical sized circular bony defect At of 2 mm diameter throughout the full cortical thickness and reaching the medullary space was performed using surgical round bur fitted on high-speed handpiece with copious irrigation of saline. Bony defects were filled manually with the tested materials according to the manufacture instructions using plastic spatula for the ion-releasing RMGI (ACTIVA) restorative material. Moreover, a stainless steel spatula was used for MTA HP and iRoot BP Plus cements, until extrusion occurred into the medullary space. Then, restorative material was light cured and cements were left to set. The bony defects in the control group were left to heal spontaneously without filling. The overlying muscles and skin were sutured with No. 0/3-resorbable suture. Floxacillin antibiotic of 1 g and ketolac tromethamine analgesic of 10 mg were given for 4 days post surgery with a dose of 0.34 mg/kg. Regular daily checkup for the physical status and the healing course of the rats was performed after surgery. Postoperatively, after one month, each rat was euthanized by administering an anesthetic overdose with ketamine hydrochloride and xylazine hydrochloride (1: 1 respectively; 0.15 mL/100 g of weight; I/P) in accordance with ISO standards 10993-1 for prolonged-permanent long-term contact of implant devices for bone tissue. The areas of interest including bone defects were surgically retrieved and prepared for routine histological investigation, SEM examination and EDX elemental analysis. All the surgical procedures were performed by the author A.A and placement of the fillings was blinded in all restorative groups, except, ion-releasing RMGI (which has a unique delivery system).
Histological specimen processing. The tibiae bones (n = 14/group) were fixed in 4% paraformaldehyde, decalcified in 10% EDTA (pH 7.4), longitudinally sectioned at the area of each bone defect. Decalcified tissues were processed in a tissue processor using ascending concentrations of ethanol, embedded in paraffin and sectioned at a thickness of 5 µm. After deparaffinization in xylene and rehydration in graded ethanol, hematoxylin and eosin (H&E) and Masson's trichrome staining were performed according to Suvarna et al. 17 , technique. Slides were photographed using Leica Microscope (Leica Microsystems GmbH, Wetzlar, Germany) adapted with a DFC420 camera under × 10 magnification to observe the amount of new bone formation, inflammatory cell response, angiogenic process, granulation tissue formation, and then under × 40 magnification to semiquantitatively estimate the number of osteoblast and osteoclast cells. The histological scoring system was performed according to the criteria described by Abid et al. 18 , with slight modification ( Table 2).The histological evaluation was performed blindly by 2 authors MG and WA. The inter-and intra-examiner reliability was verified by using Kappa test with mean percentage agreement of 90%.
Specimen processing and morphological analysis using SEM and EDX. The retrieved specimens (n = 14/group) were fixed in a solution containing 2.5% glutaraldehyde and 2% paraformaldehyde and then stored for 24 h overnight at 4 °C. The specimens were precisely cut into thin sections using ISOMET 4000, crossing the operation area and passing through the material, to expose the interface with surrounding bone for micromorphological surface analysis. The prepared disks were then washed three times for 15 min with phosphate buffered saline (pH 7.4) and fixed again in a solution of 1% sodium phosphate buffer osmium tetra oxide for 90 min. The exposed specimens' surfaces were subjected to wet polishing using 600-, 800-, 1200-, 1600-, www.nature.com/scientificreports/ 1800-and 2000-grit SiC paper, cleaned using ultrasonic cleaner containing deionized water for 30 s. Finally, the specimens underwent graduate dehydration with ethanol. The specimens were dried and coated with gold (SPI Sputter coater, USA). Examination of the processed disks is performed using JEOL JSM 6510LV scanning electron microscope (Tokyo, Japan). Energy dispersive X-ray analysis (EDX) for elemental microchemical determination was performed randomly for all specimens at the same magnification for the same areas with approximately fixed scanned area to evaluate the relative element content. EDX microanalysis weight was performed on bone material interface in selected frame to analyze entire areas and the elements were calculated from the data to evaluate the degree of mineralization of the newly formed bone. EDX element mapping was performed at the same area to detect the element distribution within the mature and newly formed bone and their presence in the surrounding tissues. Element mapping was performed through the bone-interface-material and were performed to detect the variation content of Ca, P, Si, Ta, Na, K, Al, Ba, Zr, C and O from the mature pre-existing bone towards the implanted material.

Results
Clinical findings. Generally, recovery of rats was observed after 4 days post-surgery as they returned to their routine activities, such as walking, grooming and routine dietary habits. The rats showed normal chewing efficiency without any weight loss or postoperative complications. In addition, stereomicroscopic pictures for the tibial bone of all groups showed an acceptable healing in the control one, a defect filled with ACTIVA restorative material with better osteointegration with the old bone and an excellent healing was noticed in both MTA HP and iRoot BP Plus groups as they showed marked disappearance of bone defects and the boundaries of osteotomy gaps revealed better osteointegration with the old bone ( Fig. 1).   Control group sections showed scanty, very thin, new bone trabeculae of immature woven type located mostly at the peripheral part of the tibial bone defects (n = 25) with greater amount of thick bands of typically organized granulation tissue of tightly packed collagen bundles were located centrally and at the periphery (n = 24). The granulation tissue had moderate inflammatory cell response (n = 24)) and profound sprouting angiogenesis (n = 24). Osteoblastic activity was noticed only in the peripheral part of the defect area (n = 20) and less likely at the central one (n = 8) whereas osteoclastic activity was completely absent at both areas (Figs. 2A and 3A, Table 3). Meanwhile, bone defects of ACTIVA group showed an empty space that was previously occupied by the restorative material with thick, newly formed, immature woven bone trabeculae presented only at the marginal area (n = 28). The boundaries of the defect area demonstrated mild inflammatory cell response (n = 28), moderate sprouting angiogenesis (n = 23), absence of granulation tissue (n = 26) with marked osteoblastic and osteoclastic activities (n = 26) (Figs. 2B and 3B, Table 3).
Bone defects of MTA HP group were partially filled with thick and newly formed woven bone trabeculae with wide marrow spaces presented centrally and at the periphery (n = 28) with little amount of mature granulation tissue at the central part (n = 21). The granulation tissue was characterized by mild inflammatory cell response (n = 24), moderate sprouting angiogenesis (n = 26). Osteoblastic (n = 28) and osteoclastic (n = 27) activities were found at the central and peripheral areas (Figs. 2C and 3C, Table 3). While bone defects of iRoot BP Plus group showed marked woven bone formation of normal trabecular structures with narrow marrow spaces presented centrally and at the periphery (n = 28) and a lesser amount of well-organized and mature granulation tissue formation with a residual material maintained in the granulation tissue. The granulation tissue was characterized  Table 3).
Regarding the parameters tested, Kruskal Wallis test revealed total significant differences between the control, ACTIVA, MTAHP and iRoot BP Plus groups (P = 0.000). Meanwhile, Mann-Whitney U test showed significant difference between control and ACTIVA groups, Control and MTA HP groups, control and iRoot BP Plus groups. ACTIVA and MTA HP groups, ACTIVA and iRoot BP Plus with no significant difference between MTA HP and iRoot BP Plus (Table 4).

Micromorphological analysis results. Bone is identified by the vascular canal and osteocyte lacunae.
After acid base challenge, osteoblastic-osteocyte lacunae, osteocyte lacuna-canalicular network and osteonal lamellar pattern on the surface of trabecular bone was clearly observed. Newly formed woven bone has grown in close approximation to the underlying biomaterials. It appears less homogenously mineralized with the presence of non-remodeled islands of mineralized bone. The boundaries between secondary osteons and interstitial bone and between individual trabecular packets are formed by cement lines, which are relatively hyper mineralized in comparison and therefore appear brighter, forming the interface between the old bone and the new bone. Ionreleasing RMGI (ACTIVA) showed an intimate contact and signs of bioactivity with incremental deposition of the newly formed bone along the interface. The representative images showed also a perfect contact with absence of gaps between newly formed bone and the residuals of MTA HP and iRoot BP plus (Fig. 4).
Elemental analysis outcome. The EDX results for the control sample representing the normal bone which known to be consists mainly of two compartments (Organic matter) represented by collagen and noncollagenous proteins resulting on the presence of both carbon (kα) and oxygen (kα) lines originally located at 0.2774 and 0.525 keV respectively.
The lesions of the control group specimens were filled with recently created trabecular bone with limited marrow spaces. EDX tests (Ca and P analysis) indicated a lower degree of mineralization. Lower amounts of Ca and P was expressed in the mapping analysis corresponded by higher amounts of O and C with wider bone marrow spaces side (Fig. 5A-C).  The histological segment observed by SEM showed the signs of bioactivity along the material-bone interface at the surgical defect with the formation of a layer of newly formed bone when Ion-releasing RMGI (ACTIVA) was put directly in contact with the sample on its medullar side (Fig. 6A-C). EDX and mapping analysis of the newly formed bone at the material-bone interface showed a lesser amount of Ca and P and increased amounts  www.nature.com/scientificreports/ of C and O representing the organic components, indicating the lower degree mineralizing ability of ACTIVA in comparison with iRoot BP plus and MTA-HP in terms of bioactivity. Interestingly a noticeable increased amount of silica was noticed at the interface between the material and inner medullary side which may represented as a constituent of the material. However, deposition of a newly formed bone was evident. In the direction from old pre-existing bone towards the material there was a sudden drop in the levels of distribution Ca and P inorganic elements which represents the decreased amount of mineralization of the newly formed bone at the interface. The histological segment observed by SEM showed the surgical defect entirely filled with the material and newly created bone filling the medullary space and showed the osteoinductive properties of MTA-HP strictly in contact with the sample on its medullar side. EDX microchemical analysis shows its constituent elements, i.e., Ca, Si, Mg, Al with Bi residues and high amount of P in the samples (Fig. 7A-C). Ca and P revealed a preexisting trabecular bone. Ca, P, lack of Bi, and traces of Si originating from the material were revealed by newly formed bone. In the pre-existing old bone, the Ca and P proportions were notably higher than in the newly developed bone, in particular in new trabeculae adjacent to the material (probably the newest bone). The highmagnification interface analysis at 250 × showed the complete interaction and integration of the newly formed trabeculae with the material and the absence of spaces was well demonstrated. In newly formed bones, no Bi or Al was found, but only at the interface.
The histological segment observed by SEM showed the surgical defect entirely filled with complete integration between material and newly created bone filling the entire medullary space and showed the superior bioactive properties of iRoot BP plus directly in contact with the sample on its medullar side (Fig. 8A-C). Its constitutive elements, i.e., Ca, Si, Zr, and traces of Ta, were seen on the material surface by EDX. Higher Ca and P material, traces of Si and lack of Na were shown by pre-existing trabecular bones. In newly formed bone intermingled with www.nature.com/scientificreports/ the material residues showed higher amount of Ca and P and decreased amounts of C and O which represents the organic components, indicating the higher mineralizing ability of iRoot BP plus in comparison to MTA-HP which is expressed by the mapping analysis. There was no interrupted continuity along the material-bone interface with total filling of the marrow spaces with newly formed trabecular bone intervened by the residue of iRoot BP plus material which is replaced by the newly formed bone represented by presence of small amounts of Ta.

Discussion
In the current study calcium silicate cements used for endodontic retrograde filling were tested against newly Ion-releasing RMGI (ACTIVA) restorative material in rat model through intramedullary bone tissue reaction by using H&E histological analysis, SEM morphological and EDX elemental analysis for their bio-functionality and osteo-inductive characteristics in a trial to mimic oral conditions. The old perception of biocompatibility was related to the disappearance of adverse tissue reaction to the tested material 19,20 . However, some materials were not found to induce any noteworthy interaction with the host living tissues 21 . These reactions which help the improvement of wound healing and granulation tissue formation. The recent description of biocompatibility is the ability of dental biomaterials to elicit favorable and beneficial reaction with oral tissues during its intended clinical usage 16,22 .
It has been found that the biocompatibility assessments produced by cell culture assays is not necessarily to be in agreement with animal in vivo biocompatibility implantation test 23 . Therefore, using animal models, like rodents and rabbits are suitable choice for testing biological, regenerative and mineralizing characteristics of new materials in less invasive and non-critical sized surgical procedures 24 . The ingredients of the new ion-releasing resin composite restorative material contain glass particles and a hydrophilic ionic resin matrix that facilitates the diffusion of fluoride, phosphate and calcium ions into the surrounding environment 3,22,25 . It was reported that complete healing occurred following retrograde resin placement with 97% success rate 26 . Retrograde filling with composite exhibited complete healing in 74% of cases in correlation to 59% of cases treated with amalgam 19,22 . These materials showed a higher degree of biocompatibility when subcutaneously implanted in comparison to the calcium silicate-based cements as it decrease the degree of inflammation with a better healing potentiality 16 .
A combination of in-vitro, ex-vivo, and in-vivo laboratory testing are essential to reveal the biological properties of the structure and composition of implanted materials. In-vivo animal models give initial perceptions about the physiological status, biomechanical interaction and the hormonal response against a newly tested materials, starting from rodents and rabbits as small models and ending by dogs and pigs as large ones 27 . Rabbits and rodent animal models are the key source for studying the biological behavior and regenerative potential in implant studies 28 . They are excellent candidate for modeling bone infections and extra oral surgical procedures to test the osteoinductive potential of allogeneic and xenogeneic bioactive materials 22,28 .
The outcome of this study showed the osteoinductive ability of MTA HP, iRoot BP and ACTIVA with biomineralization and new bone formation around the materials which filled the medullary space of rat tibia. Moreover, inflammation with subsequent necrosis of the cortical bone in contact with the materials was absent. In the analysis process SEM and EDX were adopted as accredited methods for the assessment of the surface morphology, the type and the amount of mineral content of the bone-material interface 29 . www.nature.com/scientificreports/ Specimen analysis showed strong co-relation between calcium and phosphorus (Ca/P) like stoichiometric hydroxyapatite. Zones of mineralization and bone-material interfacial mapping were marked by SEM imaging and EDX mapping; therefore, the differentiation between the old per-existing bone and newly formed bone and cortical and cancellous bone is explained by the differences in minerals constituents expressed by the EDX elemental analysis.
The areas of remodeling process and newly formed bone are expressed by the decreased amount of Ca and P ratios in some certain trabecular segments, while, increased amount of Ca and P with the decrease of C, Si and O indicates the directional profile from mineralizing interface towards the old mature bone.
EDS, EDX, EDAX, or EDXA simply known as Energy-dispersive X-ray spectroscopy representing a direct and powerful tool that enables scientists to analyze and identifying chemical composition of studied samples depending on the core electron ejection with high energy X-ray radiation (Moseley's Law) gives a direct correlation between frequency of emitted radiation and the atomic number of the atom 30 .
The data generated by EDX analysis consist of spectra showing peaks corresponding to the elements making up the true composition of the sample being analyzed. In bone, the most frequent application of EDX is measurement of Ca and P content in the extracellular matrix with the calculation of Ca/ P ratio. Local nanomechanical properties have been shown to correlate with Ca levels. Detection of Ca and P along the bone-biomaterial interface confirms the formation of new bone in direct contact with the biomaterial surface. EDX-mapping was performed, because this characterization method is considered an accurate method for detecting elemental distribution among the observation specimen. www.nature.com/scientificreports/ Histological examination showed that intramedullary mediated bone new formation, osteoblastic differentiation and angiogenic proliferation were implanted by MTA HP, iRoot BP and ACTIVA. Both MTA HP and iRoot BP materials showed strong biocompatibility, as only mild or moderate inflammatory infiltrate was evident quite close to the interface. While ACTIVA showed the least mineralization ability. This might be attributed to its high resinous content which countered its claimed bioactivity.
The study demonstrated the ability of an Ion-releasing RMGI (ACTIVA) and hydraulic calcium silicate cements to form osteoid matrix with activation of osteoblasts and achieving a direct bond between the materials and the surrounding bone matrix. The formation of favorable environment by calcium silicate cements for osteoblastic differentiation with new bone formation through interfacial active biological ions exchange and its effect on cellular activity could explicate the (bio-mineralizing) response of the tested materials. Bioactivity of a material is related to its chemical reactivity with living tissues through favorable interfacial ion exchange and precipitation reactions 31 , and for biomineralization by induction for osteogenic extra-cellular signaling with summoning cascades of cellular differentiation into osteoblasts with osteoid matrix lay down at the surgical site 32 .
Calcium silicate cements shown in a previous studies to have the ability to provide high alkaline pH in the media and release calcium ions after formation of hydrated silica-rich layer 33 . Therefore, generating a favorable environment at the interface with continuous provision of biologically active ions through the surface for apatite deposition 34 . In addition, silica Si ions as a byproduct has a role in new bone formation as it encourages the angiogenesis through elevating gene expression which is synergetic with the differentiation of osteoblasts and extracellular matrix synthesis and deposition 35 .
The setting mechanism of calcium silicate cements starts after hydration by water during the mixing process. The formed calcium silicate hydrate by-products formed after mixing have many phases as reported by Gandolfi et al 36 . These stages including porous colloidal CSH gel and radial acicular CSH crystals, rhombohedral crystals of portlandite, needlelike crystals of ettringite which is a hexacalcium alumina-tetrisulphate hydrate, and calcium mono-sulfoaluminate or calcium mono-carboaluminate. Moisture plays a major role in initiation of apatite formation (bioactivity) and nucleation process of calcium ions on the surface of hydrated calcium silicate particles, it goes through defined stages starting from rapid dissolution of calcium ions after the solid-liquid interface formed on the cement particles forming portlandite. A porous, fine-grained/fibrous and disorganized hydrated silicate gel layer is formed by alkaline etching; containing Si-OH silanol groups and negative surface charges that may act as nucleation sites for apatite formation. Portlandite crystals nucleate inside the hydrating wet paste due to a strong continuous outflux of calcium hydroxide from CSH that occurs during the first hours after mixing. Calcium phosphate and apatite deposit on cement surface as result of exposure to tissue fluids after being placed in vivo 37 . Calcium hydroxide formed reacts with carbon dioxide in the tissues to form calcified nodules which acted as nucleus for mineralization 38 . In addition, Zamparini et al. 39 revealed that ready-made paste bioceramic materials containing tantalum pentoxide as radiopacifier showed higher calcium and silicate ions release.
The hydrophobic nature of resin component in light-cured calcium silicate containing dental materials make them less soluble than MTA. The pores found in the resin matrix which allow for water penetration with inward outward flux, therefore it could explain their capability of releasing of calcium through solubilization of calcium ions and calcium silicate hydrate and portlandite formation 40 . Morrow et al. 41 compared the calcium releasing ability of ACTIVA, Dycal and TheraCal in 7 days period, results showed that ACTIVA released an amount of 0.72 μg/mm 2 which was less than that released from Dycal (4.88 μg/mm 2 ) and TheraCal 9.12 μg/mm 241 .
In the context, it has been reported that hydroxy apatite particles formed on the surface of ACTIVA after 30 days of immersion in phosphate buffered saline after utilization of SEM, PulseTor SDD (Silicon Drift Detector) and Energy-dispersive X-ray spectroscopy (EDS) analysis 42 . It has been also shown that ACTIVA has the ability to form dentinal tubules integrated resin tags with 2.0-2.5 Ca/P ratio across the hybrid layer 3,42 .
Actually, in the present study ACTIVA showed promising biomineralizing response but the tested calcium silicate cements showed better performance in biomineralization, angiogenic activity with neovascularization and formation of capillaries close to the materials. In addition, MTA HP and iRoot BP were incorporated with surrounding bone showing intervening between old and newly formed bone without recognition of the materials exhibiting no interposed connective tissue, resorption lacunae inflammatory response, rejection or necrosis in the adjacent pre-existing bone and medullary side. No osteoclastic activity or multinucleated giant cells observed in the histologic analysis except for a few monocytes and lymphocytes existed. It can be assumed that the high alkaline pH played a significant role in inactivation of osteoclasts as explained in previous studies 43,44 , and previous study demonstrated the high alkaline pH of iRoot BP and MTA HP and nearly neutral pH for ACTIVA 45 .
In light of this study outcome, the null hypothesis was rejected. A part of the laboratory work of the study was performed during Covid-19 pandemic time and our research team faced some delay and shifting from the original timeframe due research laboratory closure. Also, further advanced radiographical methods (such as Micro-CT) is recommended in future studies.

Conclusion
Calcium silicate-based cements (MTA HP & iRoot BP Plus) and resin-modified glass ionomer restorative material (ACTIVA) exhibit the ability to induce new bone formation through osteo-inductivity. The tested resin-modified glass ionomer restorative material does not induce fibrous encapsulation or any foreign tissue reaction with the direct contact between material superficial layer and mineralized bone matrix.

Data availability
All datasets generated or analysed during this study are included in this published article [and its Supplementary  Information